【作者】 陈阳；

【导师】 张积森；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 甘蔗是全世界最重要的糖料和能源作物。在自然栽培条件下,病虫害是影响甘蔗产量的重要因素。目前世界上已知的甘蔗病害已达160多种,我国发现的有60多种。其中大陆蔗区发生的大约50种,常见的甘蔗病害有宿根矮化病、凤梨病、线虫病等。一般为了控制植物疾病的发生和提高作物产量,人们会直接采用喷洒农药的方式,但农药的使用不仅会使病原体的耐药性增强,而且药物的累积,会造成土壤、水源以及空气污染,最终会对人体健康造成威胁。植物本身就有一系列抵御外界病原体侵害的系统,植物疾病抵抗基因可以编码抵抗蛋白用来阻止外源病原体入侵。这类基因中最为重要的便是NBS-LRR基因。现代甘蔗栽培种是由热带种和割手密种杂交而来,这些杂交栽培种的高糖特性来自于热带种,而抗病虫害等抗逆性的遗传属性来自于割手密。因此本研究选择抗逆性和抗病性较强的甘蔗割手密种为研究材料,综合应用生物信息学手段对甘蔗割手密种全基因组的NBS-LRR基因家族进行分类与演化分析,并与单双子叶代表性的植物,拟南芥、菠萝、无油樟、水稻、玉米和高粱进行聚类分析与结构比较。研究的结果将为甘蔗抗病基因的分子机制和分子育种提供重要的理论参考。本研究获得的主要结果如下:(1)应用比较基因组手段,以水稻等模式植物的NBS-LRR基因组数据为参考,在项目组已有的甘蔗基因组数据基础上,利用HMM结合Pfam数据库鉴定出具有甘蔗NBS共506个。根据基因的功能结构域将这些NBS分为四个类型,其中 242 个为 CC-NBS-LRR、120 个为 NBS-LRR、113 个为 CC-NBS、31 个为NBS,总数量和水稻接近。在NBS类型基因中,数量最多的是NBS-LRR家族为362个,因此推测该基因家族曾发生基因复制事件。(2)应用MEME对甘蔗不同类型的NBS序列的基序进行分析,鉴定结果表明甘蔗NBS结构域的基本基序都在,值得注意的是MHDV基序在甘蔗中为VHDM/MHDL/VHDL,GLPL 则部分为 GVPL。(3)根据甘蔗NBS蛋白质序列构建系统发育树,四种类型的NBS基因都交错分布在系统发生树上,这表明了甘蔗的NBS基因的各个类型基因属于平行起源。(4)运用比较基因组学,将甘蔗与无油樟、拟南芥、菠萝、玉米、水稻、高粱进行系统发生树构建。在高粱和玉米的NBS基因中并未发现TIR结构域,且TIR结构域在单子叶植物中极少存在,大部分都消失。这证实了 TNLs在单子叶植物和双子叶植物分化之前消失或者退化。从不同植物的NBS基因数量与基因组大小进行比较发现,基因组大小并不直接决定了 NBS基因数量的多少。基因组大小为2500 M的玉米,其NBS基因仅有107个,这个数量比基因组125 M的拟南芥的数量还少,而葡萄的基因组大小仅为475 M,其NBS类型的基因就有535个。(5)IAA、ABA、乙烯、GA等激素是植物的生物和非胁迫相关的信号,通过RNA-Seq技术对甘蔗NBS-LRR基因家族的362个基因的FPKM值在激素处理后的甘蔗割手密种中的表达水平进行分析。值得关注的是,在CC-NBS-LRR亚家族中发现一个特别的基因AP850660980,该基因在IAA处理下的表达量与其它三种激素不同,当在IAA激素处理下达到最高表达量时,其它三种激素表达量在激素处理前后不发生改变,而当其它三种激素表达量同时达到最高值时,IAA处理的实验组表达量不改变。因此推测在该基因的表达中IAA和ABA,乙烯,GA三种激素存在互补关系。在NBS-LRR亚家族的表达谱中也发现了一个比较特别的基因,AP851288080,该基因在GA和IAA处理的表达谱中,表达趋势基本相同,就连最高表达值和最低表达值的时间段和组织部位都相同,有可能该基因在对这两种激素的应答机制较为相似的原因。更多还原

【Abstract】 As an important sugar-yielding crops,sugarcane also suffered the plant disease which caused by the virus,bacteria,fungi and nematodes as well.Besides artificial control,plant has its defense mechanisms to resistant the pathogens.Plant disease resistance genes(R genes)encoding resistance proteins which has function for preventing the invasion of exogenous pathogen.The genes encoding nucleotide binding site and leucine-rich repeat proteins(NBS-LRR genes)comprise the largest class and account for more than half of R genes in plant,and were revealed to play a critical role in defending plants from a multitude of pathogens and pests.Modern sugarcane were derived from the crosses between S.officinarum and S.sponteaneum.High sugar content trait of hybrid cultivars was suggested to be contributed by S.officinarum,while the stress tolerance was attributed by S.spontaneum.In this study,combined comparative genomics and whole genome sequences resources of S.spontaneum,the whole set of NBS-type genes were identified and characterized from S.spontaneum.Phylogenic and gene structure analysis were used to study the evolutional relationship of NBS-LRR gene families in S.spontaneum and angiosperm.Moreover,the gene expression profiles of NBS-LRR gene families were investigated for evaluating the gene functions in S.spontaneum.The main results are as follows:(1)Based on HMM and Pfam database,a total of 506 genes were identified to be the NBS class of genes in S.spontaneum species.The functional domain analysis revealed that the NBS family can be divided into four types with different domain organizations,including 242 Coiled-coil NBS-LRR genes,120 NBS-LRR genes,113 Coiled-coil-NBS and 31 NBS genes.(2)With the MEME analysis,eight conserved motifs(P-LOOP,GLPL,Kinase,RNBS-A,RNBS-B,RNBS-C,RNBS-D,MHDV)of plant NBS type genes were also found from the four types of NBS-encoding genes.Among the eight motifs,GLPL,MHDV,P-LOOP and Kinase-2 are the top four conserved motifs of NBS domain in different subfamily of sugarcane.Noteworthy,in sugarcane,instread of MHDV,VHDM or MHDL were presented in NBS-LRR of sugarcane.In addition,RNBS-A,RNBS-B and RNBS-C are usual presented in NBS domain,while,RNBS-C was absent both NBS-LRR subfamily and NBS subfamily in sugarcane.(3)Similar to previous study in monocotyledons,TIR domain was absent in NBS families in sugarcane.Based on the gene number comparisons among the examinated plant species,there were no correlation between the monoploidy genome size and the numbers of NBS-LRR genes in plant.(4)Comparative analysis of gene structures and phylogenetic evolution were performed among arabidopsis,pinapple,rice,maize,sorghum and sugarcane.Phylogenetic analysis showed that the four type of NBS-LRR genes were not group together,suggesting that the four subfamilies were originated before the last common ancestor of NBS-LRR families.The phylogenetic distribution of sugarcane NBS resistance genes was consistent to that of sorghum,demonstrating that there were no gene gain/loss event after the divergence of sorghum and sugarcane.(5)To investigate the expressional level of NBS-LRR genes in S.spontaneum,we performed comparative transcriptome profiling at different tissue under four hormone treatments(ABA,Et,GA,IAA).Noteworthy,a CC-NBS-LRR gene AP850660980 presented in opposite directions for the gene expression profiles between IAA treatment and the other three kinds of hormones.The gene has the highest expresional epxression under the treatment of IAA at 96 hours in stem,while,showed no variation under the other three hormone treatments;in contrast,the gene presented no variation under the treatment of IAA at 24 hours in stem,while,reached the highest under the other three kinds of hormone.Thus,it is speculated that there is a complementary relationship between IAA and the other three hormones for regulating the expression of the gene.In addition,NBS-LRR gene AP851288080 was also deserved to be paid attention.This gene have consistent expressional profiles under the treatment of GA and IAA,indicating that the genes has the similar cross-talk mechanism under these two kinds of hormone.Our study is the first report for the whole gene families of NBS-LRR in S.spontaneum,providing an important basis for further investigating the molecular mechanism of the disease-resistant and assistant the molecular breeding in plants.更多还原